Cannabidiol inhibits both human K_V7.1 and K_V7.1/KCNE1 channels through distinct sites

Several essential physiological systems express voltage-gated potassium channels within the K_V7 family (comprising K_V7.1-7.5), sometimes also co-assembled with auxiliary subunits in the KCNE family (comprising KCNE1-5). An ongoing challenge to K_V7 drug development is creating subtype-selective compounds to limit adverse effects. Prior work has shown that the antiepileptic cannabidiol (CBD), a pan-K_V7 modulator, inhibits cardiac- and epithelia-associated K_V7.1 and K_V7.1/KCNE1 channels, while activating neuronal K_V7 subtypes (K_V7.2-7.5). However, little is known about the binding sites through which CBD mediates inhibitory effects on K_V7.1 and K_V7.1/KCNE1, limiting insight towards the development of selective K_V7 modulators. To address this knowledge gap, we used a combination of the Chai-1 artificial intelligence model (to generate CBD binding site predictions in human K_V7.1 and K_V7.1/KCNE1 channels), site-directed mutagenesis and electrophysiology of these channels expressed in Xenopus laevis oocytes (to corroborate CBD binding site predictions), and molecular dynamics simulations (to study the biophysical mechanisms underlying CBD binding). We found that CBD binds to two unique sites within K_V7.1 and K_V7.1/KCNE1. In K_V7.1 alone, CBD was bound to an intrasubunit S5-S6 pore domain binding site; referred to as the S5-S6 site. In K_V7.1/KCNE1, the addition of the KCNE1 subunit created a novel binding site for CBD, sandwiched between two K_V7.1 subunits and one KCNE1 subunit; referred to here as the S6-S5'-E1 site. Molecular dynamics simulations showed that CBD binding to the S6-S5'-E1 K_V7.1/KCNE1 site closes off the K_V7.1 S5-S6 site. A sequence comparison between K_V7 channels revealed key amino acid differences at both the S5-S6 and S6-S5'-E1 sites relative to neuronal K_V7s. These support the notion that CBD binds differently in K_V7.1 and K_V7.1/KCNE1 channels in accordance with its unique inhibitory pharmacological effects on these channels compared to the activating effect in neuronal K_V7s. Thus, we provide support for K_V7.1 and K_V7.1/KCNE1 being inhibited by CBD via distinct binding sites, which can guide future research focused on the rational development of drugs that avoid inhibitory effects on K_V7.1 and K_V7.1/KCNE1 channels or utilize these sites to modulate channel activity.